Quantitative Characterization of Adipocyte Subtypes

SGBS cells were plated on eight-well ibidi micro slides (Ibidi GmbH, Germany) and differentiated as previously described. Immunofluorescence staining was carried out as described previously^{27 (link)}. Scanning was done by an iCys Research Imaging Cytometer (iCys, Thorlabs Imaging Systems, Sterling, VA, USA). The images were processed and analyzed (n = 3, 2000 cells per SGBS sample) by our high-throughput automatic cell-recognition protocol^{27 (link)} with some modifications using the iCys companion software (iNovator Application Development Toolkit version 7.0, CompuCyte Corporation, Westwood, MA, USA). As previuosly, cells were identified according to their Hoeschst 33342 nuclear staining. Then, the fluorescence signal intensity of the UCP1 immunostaining and the texture sum variance of the light scatter signal of lipid droplets were quantified in each cell within the 30-pixel immediate outward vicinity of the nucleus contour by the Cell Profiler software (The Broad Institute of MIT, MA, USA). Afterward, based on these fluorescence and light scatter signal of single cells, a semi-automated classification and enumeration of the differentiated white and brown adipocytes and undifferentiated preadipocytes was carried out applying the trained classification “Fast Gentle Boosting” of the Cell Profiler Analyst software (The Broad Institute of MIT, MA, USA).